U2AF1 S34F enhances tumorigenic potential of lung cells by exhibiting synergy with KRAS mutation and altering response to environmental stress

SUMMARY Although U2AF1 S34F is a recurrent splicing factor mutation in lung adenocarcinoma (ADC), U2AF1 S34F alone is insufficient for producing tumors in previous models. Because lung ADCs with U2AF1 S34F frequently have co-occurring KRAS mutations and smoking histories, we hypothesized that tumor-forming potential arises from U2AF1 S34F interacting with oncogenic KRAS and environmental stress. To elucidate the effect of U2AF1 S34F co-occurring with a second mutation, we generated human bronchial epithelial cells (HBEC3kt) with co-occurring U2AF1 S34F and KRAS G12V . Transcriptome analysis revealed that co-occurring U2AF1 S34F and KRAS G12V differentially impacts inflammatory, cell cycle, and KRAS pathways. Subsequent phenotyping found associated suppressed cytokine production, increased proliferation, anchorage-independent growth, and tumors in mouse xenografts. Interestingly, HBEC3kts harboring only U2AF1 S34F display increased splicing in stress granule protein genes and viability in cigarette smoke concentrate. Our results suggest that U2AF1 S34F may potentiate transformation by granting precancerous cells survival advantage in environmental stress, permitting accumulation of additional mutations like KRAS G12V , which synergize with U2AF1 S34F to transform the cell.


INTRODUCTION
Splicing factor mutations are prevalent in cancer and lead to global dysregulation of RNA splicing in protein-coding genes 1,2,3,4,5,6,7,8,9,10 .Work still remains to fully characterize the functional consequences of dysregulated splicing.U2AF1 is among the most significantly mutated genes in lung ADC and codes for a splicing factor 11 .Of the U2AF1 mutations in lung ADC, U2AF1 S34F occurs the most frequently 2 .U2AF1 is a subunit of the U2 Auxiliary Factor complex 12 .Wild-type U2AF1 (U2AF1 WT ) facilitates spliceosome assembly by recognizing and binding to the 3' splice site 13 .U2AF1 can also directly bind to mRNA to repress protein translation 14 .In U2AF1 S34F cells, the amino acid substitution in the second zinc finger alters 3' splice site choice 14,15,16 and creates isoforms which result in abnormal gene expression 17 .Other impacts of this mutation include altered binding to mRNA leading to translational dysregulation, We obtained two parental isogenic HBEC3kt clones that were either wild-type or mutant for U2AF1 15 : one cell line was homozygous U2AF1 WT , and the other cell line was heterozygous for U2AF1 S34F at its endogenous locus (Figure S1 A).Homozygous U2AF1 S34F mutation is lethal, so was not a consideration 27 .A KRAS G12V pLenti6_V5 plasmid 25 has previously been used to identify genetic perturbations required to accomplish in vivo transformation of HBEC3kts, which we obtained to study the impact of co-occurring U2AF1 S34F and KRAS G12V mutations on preneoplastic potential.We exogenously overexpressed KRAS G12V in U2AF1 WT and U2AF1 S34F HBEC3kt cells using this construct.As a transduction control, we also introduced LacZ using the same plasmid backbone.A total of 4 cell lines were generated per parental HBEC3kt clone: (1) U2AF1 WT + LacZ (2) U2AF1 S34F + LacZ, (3) U2AF1 WT + KRAS G12V , (4) U2AF1 S34F + KRAS G12V .Lentivirus is known to unpredictably integrate into the host genome, and other groups' use of this KRAS G12V vector reported variation in KRAS G12V expression 25 .The presence of U2AF1 S34F and KRAS G12V was validated through transcript expression and immunoblot (Figures S1 B-F).Our immunoblot and gene expression analysis revealed that KRAS overexpression was inconsistent across the cell lines in clone 1 (Figures S2 A-E).
Using these four cell lines, we performed short-read RNA sequencing and cell-based assays to understand how U2AF1 S34F and KRAS G12V co-occurrence alters the transcriptome and biology of HBEC3kt (Figure 1B).We performed differential gene expression and gene set enrichment analysis on RNA-seq data (Figure 1C, Table S2).A gene set uniquely downregulated in U2AF1 S34F + KRAS G12V HBEC3kts is the KRAS Signaling Down gene set, corresponding to genes downregulated when KRAS is active 28 .In contrast, the U2AF1 WT + KRAS G12V cell line had a positive enrichment for the KRAS Signaling Up gene set, corresponding to genes upregulated when KRAS is active, and no significant enrichment in the KRAS Signaling Down gene set.From this result, we infer that U2AF1 S34F in the presence of oncogenic KRAS alters the KRAS signaling pathway.
We also observed that individually, U2AF1 S34F and KRAS G12V produced opposite enrichment patterns to each other in KRAS signaling, coagulation, and inflammatory pathway gene sets (KRAS Signaling Up, Coagulation, IL6 JAK STAT3 Signaling, Inflammatory Response, TNFA Signaling Via NFKB, Interferon Alpha Response, Interferon Gamma Response).In some gene sets (Complement, IL2 STAT5 Signaling, Unfolded Protein Response, Inflammatory Response, TNFA Signaling Via NFKB, Interferon Alpha Response, Interferon Gamma Response, E2F Targets, and G2M Checkpoint), the U2AF1 S34F enrichment pattern persisted in the U2AF1 S34F + KRAS G12V cell line.In others (KRAS Signaling Up, Coagulation, IL6 JAK STAT3 Signaling), the opposite enrichment patterns caused by either mutation alone appeared to counter each other in the U2AF1 S34F + KRAS G12V line, producing nonsignificant enrichment in the gene sets.This suggests that KRAS G12V suppresses U2AF1 S34F -specific gene expression signatures in the U2AF1 S34F + KRAS G12V cell line.To understand this observation in the context of expression patterns in patient-derived samples, we quantified the ratio of U2AF1 S34F mRNA in cells with and without KRAS G12V using our short-read RNA-seq data.A subset of lung ADC primary samples have been reported to have "quasi-WT" status, which represents tumors with low S34F:WT mRNA ratios, but unchanged absolute U2AF1 S34F or total U2AF1 mRNA levels 15 .These quasi-4 WT S34F:WT mRNA ratios range from 0.27-0.31.We found that the U2AF1 S34F mRNA fraction in U2AF1 S34F + KRAS G12V cells falls within the range of 0.26-0.37(Figure 1D, Table S1) compared to the range of 0.41-0.54when U2AF1 S34F is present alone.This suggests that the presence of KRAS G12V may suppress U2AF1 S34F expression to the levels seen in "quasi-WT" patient samples.
We then examined the mutational status of typical-S34F and quasi-WT samples from The Camcer Genome Atlas (TCGA) lung ADC cohort studied by Fei et al. 15 .Consistent with our hypothesis that mutant KRAS suppresses U2AF1 S34F expression signature, we found that quasi-WT patient samples carry a higher proportion of KRAS mutations (3/4 samples) than typical-S34F samples (5/9) (Figure 1E), although the difference is not statistically significant, likely due to low sample sizes.Together, these results support the hypothesis that KRAS G12V suppresses the U2AF1 S34F transcriptomic signature.
We also found variation in the U2AF1 S34F mRNA ratios between isogenic clones (Figure S2 F, Table S1).The U2AF1 S34F mRNA fraction of clone 1 U2AF1 S34F + KRAS G12V ranged from 0.31-0.37,while the U2AF1 S34F mRNA fraction of clone 2 U2AF1 S34F + KRAS G12V ranged from 0.26-0.28.Although clone 1 ranged higher in U2AF1 S34F mRNA ratio than clone 2, it was still well within the typical-S34F range reported by Fei et al. (0.43 or above) 15 .This result, along with our immunoblot of KRAS G12V and KRAS gene expression analysis, indicated that inconsistent KRAS G12V integration in clone 1 impacts both protein abundance and the transcriptome.Additionally, oncogenic KRAS has been shown to alter splicing 26 .Thus, we continued subsequent transcriptomic analyses and the majority of our phenotypic experiments interrogating the interaction of U2AF1 S34F and KRAS G12V with clone 2.
We detected and quantified splicing events from short-read data using JuncBASE 32 (See Data Availability).As expected, U2AF1 S34F + LacZ HBEC3kts exhibited the most changes in differentially spliced genes, compared to U2AF1 WT + LacZ (Figure 2A).U2AF1 WT + KRAS G12V HBEC3kts displayed the lowest amount of differentially spliced genes (Figure 2B), while U2AF1 S34F + KRAS G12V HBEC3kts displayed an intermediate number (Figure 2C).Consistent with the effect that KRAS G12V has on expression of U2AF1 S34F , these results suggest that KRAS G12V suppresses the splicing changes mediated by U2AF1 S34F .
We next compared the categories of splicing events that were significantly different (adjusted p < 0.25 and |∆PSI| ≥ 10) between U2AF1 S34F + LacZ and U2AF1 WT + LacZ, and U2AF1 S34F + KRAS G12V and U2AF1 WT + LacZ.JuncBASE categorizes splicing events in eight different 5 categories: cassette exon, mutually exclusive exon, coordinate cassette exons, alternative 5' splice site, alternative 3' splice site, alternative first exon, alternative last exon, and retained intron (Figure 2D, Table S1).We found that co-occurring U2AF1 S34F and KRAS G12V mutations had a similar fraction of cassette exon events as U2AF1 S34F alone, a splicing event type characteristic of U2AF1 S34F 2,14,15,17,30,31 .This persistence of U2AF1 S34F -specific splicing event was consistent with our gene expression results, which demonstrated that certain U2AF1 S34Fspecific enrichment patterns persisted in the U2AF1 S34F + KRAS G12V line.In contrast, the alternative first exon and alternative last exon events in U2AF1 S34F + KRAS G12V HBEC3kts appeared to be at an intermediate fraction between those in U2AF1 S34F + LacZ and U2AF1 WT + KRAS G12V cells.Similar to gene expression enrichment patterns, we hypothesize that the effects of co-occurring U2AF1 S34F and KRAS G12V mutations on splicing antagonize with each other to create intermediate splicing events proportions.
To examine potential biological pathways impacted by the differentially spliced genes, we performed gene set enrichment analysis (GSEA) on differentially spliced genes.In contrast to the differential gene expression GSEA results, far fewer Hallmark gene sets were significantly differentially enriched amongst our genotypes.Only one gene set, p53 Pathway, was found to be significantly enriched, and only in the U2AF1 S34F + KRAS G12V comparison.This finding highlights the non-overlapping roles of gene expression and splicing on transcripts belonging to certain pathways in the cell.
A recently appreciated role of U2AF1 S34F is its ability to confer resistance to the effects of stress.U2AF1 S34F alone has been shown to increase cell proliferation following ionizing radiation exposure 14 .U2AF1 S34F has also recently been found to confer altered splicing and binding to stress granule gene sets in an MDS cell line 21 .Stress granules are RNA-protein condensates that may help cells survive stress and can help cancer cells resist chemotherapy 33 .In the MDS line, altered splicing in stress granule genes was associated with enhanced viability under sodium arsenite, a chemical agent of oxidative stress 21 .
We hypothesized U2AF1 S34F may also be altering stress response through aberrant splicing in our cell lines.We observed increased splicing in stress granule protein genes in U2AF1 S34F + LacZ, compared to other genotypes (Figure 2E, Data S1) Interestingly, gene expression changes in in cells with U2AF1 S34F background also showed increased expression in this gene set compared to cell lines without U2AF1 mutation (Figure S3 A, Table S2).

Co-occurring U2AF1 S34F and KRAS G12V mutations increase oncogenic potential and proliferation
Following this transcriptomic profiling, we sought to understand the functional consequences of differentially expressed gene sets.We first sought to explore gene sets with similar enrichment patterns in both U2AF1 S34F + LacZ and U2AF1 S34F + KRAS G12V HBEC3kts, as they indicated U2AF1 S34F -specific effects which persisted when KRAS G12V was present.One category that fit this criteria was the inflammatory pathway gene sets (Complement, IL2 STAT5 Signaling, IL6 JAK STAT3 Signaling, Inflammatory Response, TNFA Signaling Via NFKB, Interferon Alpha Response, and Interferon Gamma Response), where the presence of U2AF1 S34F was associated with downregulation.Oncogenic Ras has been found to increase production of cytokines such as IL-6 in multiple cell types 34,35 .To probe how these pathways are altered in our U2AF1 S34F + KRAS G12V cell line, we measured inflammatory cytokine production in our HBEC3kt cell lines.For most cytokines tested, we observe that U2AF1 WT + KRAS G12V HBEC3kts secrete the highest levels of inflammatory cytokines.Co-occurrence of U2AF1 S34F with KRAS G12V suppresses the levels of secreted cytokines IL-1β, IL-6, IL-8, TNFα, GM-CSF, and IFNγ (Figure 3A, Table S1).High levels of IL-1β, TNFα, GM-CSF, and IFNγ have been shown to promote antitumor activity in animal models 36,37,38,39 .We hypothesized that U2AF1 S34F creates a microenvironment conducive to tumor growth by bringing cytokine secretion down to an intermediate level in KRAS G12V -mutant cells.We note that our cytokine results are inconsistent with previous work done on U2AF1 S34F HBEC3kts, which showed that U2AF1 S34F increases the secretion of cytokines such as IL-8 14 .However, the clonal background of the cells used in the aforementioned study was not reported, and it is possible that different steady-state cytokine secretion levels may be present in HBEC3kts from different isogenic clones.
HBEC3kts with U2AF1 S34F also exhibited expression in gene sets related to cell cycle progression (E2F Targets, G2M checkpoint).To understand how these gene expression differences translate to altered phenotype, we next asked how U2AF1 S34F and KRAS G12V mutations affect proliferative potential.We stained HBEC3kts with EdU and phospho histone-H3 (PHH3) to measure the proportion of cells undergoing S-phase and M-phase, respectively 40,41 (Figures 3B-C, Table S3).Previous models with U2AF1 S34F have found that U2AF1 S34F by itself suppresses growth phenotypes such as proliferation and colony-forming potential 15,22 .Consistent with these findings, we observed lower normalized EdU and PHH3 intensity in U2AF1 S34F + LacZ HBEC3kts compared to U2AF1 WT + LacZ.However, when KRAS G12V and U2AF1 S34F co-occur, we observe increased proliferation compared to U2AF1 S34F by itself.Notably, M-phase staining in U2AF1 S34F + KRAS G12V HBEC3kts was elevated to above U2AF1 WT + LacZ levels (Figure 3C), indicating that KRAS G12V confers increased mitosis in U2AF1 S34Fmutant cells.
Mutant KRAS, including KRAS G12V is known to induce oncogenic phenotypes, such as anchorage-independent growth 42 .Due to the enhanced proliferation in U2AF1 S34F + KRAS G12V cells and the unique negative enrichment score in the KRAS Signaling Down gene set observed in this line, we hypothesized that co-occurring U2AF1 S34F and KRAS G12V would alter anchorageindependent growth as well.We cultured HBEC3kts of the four genotypes on low attachment plates and measured viability.U2AF1 S34F + KRAS G12V HBEC3kts survived anchorageindependent growth conditions better than other genotypes over 10 days in low-attachment conditions (Figure 3D, Table S1).
Finally, we sought to understand how U2AF1 S34F and KRAS G12V co-occurrence in HBEC3kts impacts the ability of cells to form tumors, in vivo.Cells from the four genotypes were injected into NOD scid gamma (NSG) immunodeficient mice.We find that U2AF1 S34F + KRAS G12V HBEC3kts formed more tumors than HBEC3kts with either mutation alone (Figure 3E).This suggests that co-occurring U2AF1 S34F and KRAS G12V mutations synergize to transform HBEC3kts cells in vivo.
Similar to the heterogeneity observed in KRAS gene expression, we also observed phenotypic heterogeneity between our isogenic clones.When we asked how the tumor formation differed between cells from clone 1 and clone 2, we found that clone 2 U2AF1 S34F + KRAS G12V HBEC3kts formed tumors more frequently than clone 1 (Figure S3 B).In contrast, the one tumor formed by a U2AF1 WT + KRAS G12V was from clone 1.Similarly, when we examined viability in low-attachment conditions, we observed that the U2AF1 S34F + KRAS G12V cell line from clone 2 was more viable in low attachment conditions at earlier timepoints than clone 1 (Figure S3 C, Table S1).Importantly, no tumors were formed with U2AF1 S34F , suggesting that U2AF1 S34F alone is insufficient for in vivo transformation.These results are consistent with the hypothesis that inconsistent KRAS G12V integration in clone 1 impacts oncogenic potential unpredictably.
We also assayed for other cancer hallmarks in clone 2, such as the long-term ability to survive and proliferate into colonies, which is a marker of cancer stemness and can be assessed with a clonogenicity or colony-forming assay 43 .Interestingly, co-occurring KRAS G12V and U2AF1 S34F suppressed colony-forming potential (Figure S3 D, Table S1).These findings are consistent with previous work performed on U2AF1 S34F -mutant cancer cell lines 22 .We also performed a woundhealing assay to assess the invasive potential of U2AF1 S34F + KRAS G12V HBEC3kts (Figure S3 E, Table S1) and observed that U2AF1 S34F decreases invasive potential in KRAS G12V background.Our results indicate that enhanced proliferation conferred by U2AF1 S34F + KRAS G12V may work in concert with pathways outside of stemness and invasion to confer oncogenic potential.

Altered splicing in stress granule genes in U2AF1 S34F HBEC3kts is associated with enhanced stress response
Stress granules are often induced by an agent of oxidative stress 21,44 .Oxidative stress is relevant to cancer formation because it can produce mutations by causing DNA damage 45 .In lung ADC patients, a common source of oxidative stress is exposure to cigarette smoke.To understand the connection between oxidative stress response and lung ADC, we next analyzed splicing alterations in primary sample data from TCGA.We found greater numbers of splicing alterations in lung ADC samples from patients with smoking histories than in never-smokers (Figure 4A and See Data Availability).Although we observed a trend of increased U2AF1 mutations in samples from patients with smoking histories, high splicing alterations were present in U2AF1 WT samples in this group as well.
Previous studies on U2AF1 S34F have observed an increase in viability after exposing cells to oxidative stress like radiation and sodium arsenite, compared to U2AF1 WT cells 14,21 .We followed up on this line of inquiry by measuring how U2AF1 S34F alone impacts viability in cigarette smoke concentrate (CSC) (Figure 4B, Table S1).Because enhanced resistance to stress appeared to be conferred by U2AF1 S34F alone, We treated U2AF1 S34F or U2AF1 WT HBEC3kts from both 8 clone 1 and clone 2 with CSC and measured viability after three days.U2AF1 S34F HBEC3kts displayed higher viability than U2AF1 WT HBEC3kts at all concentrations tested.Together, our results lead us to a model of oncogenic transformation.U2AF1 S34F has been reported to be a truncal mutation in lung cancer and MDS 16,23,46 .We propose that U2AF1 S34F , when present in precancerous cells, allows cells to survive an initial onslaught of oxidative stress better.The surviving cells are more likely to persist and accumulate further mutations like KRAS G12V , which act synergistically with U2AF1 S34F to alter splicing and gene expression, resulting in an increase in oncogenic potential (Figure 4C).

DISCUSSION
Despite its status as a recurrent mutation, the role of U2AF1 S34F in lung cancer has been difficult to understand since the mutation confers anti-proliferative and anti-invasive phenotypes when present alone in model systems.This aspect limits the ability of researchers to identify the functional role of U2AF1 S34F in lung cancer and limits the use of U2AF1 S34F as a prognostic marker for lung ADC.To gain a better understanding of this mutation, we examined the role of U2AF1 S34F in early cancer formation in two directions: how U2AF1 S34F may synergize with other cancer drivers like KRAS G12V , and how U2AF1 S34F by itself can impact stress response in the cell.
Validation of pathways predicted to be differentially altered by gene set enrichment analysis revealed that the direction of enrichment in a gene set did not always correspond with the direction of pathway change in phenotype.For instance, although cell cycle gene sets were both positively enriched in U2AF1-mutant cell lines regardless of KRAS status, U2AF1 S34F + LacZ cells exhibited reduced proliferation.In contrast, the co-occurrence of U2AF1 and KRAS mutations increased proliferation.Other gene set enrichment patterns translated to more consistent phenotypes.For instance, a negative enrichment in inflammatory gene sets translated to suppression of inflammatory cytokines for HBEC3kt lines.
We also examined splicing-level changes in the transcriptome caused by U2AF1 and KRAS mutations.Interestingly, we found little overlap in gene set enrichment between differentially expressed and differentially spliced genes, highlighting the importance of using multiple kinds of RNA-seq analysis to assess the synergistic impact of mutations.For example, we observed increased alteration in stress granule protein genes unique to samples harboring U2AF1 S34F in both splicing analysis and differential gene expression results.We also detected U2AF1 S34Fspecific splicing event types, such as cassette exon event usage, that persisted in U2AF1 S34F + KRAS G12V cells, while KRAS G12V presence suppressed other U2AF1 S34F -specific event types to an intermediate level.
When we followed up on our splicing analysis by quantifying cellular stress response, we found that U2AF1 S34F confers resistance to exposure to cigarette smoke concentrate.Our work leads us to a model in which U2AF1 S34F confers oncogenic potential that is dependent on the presence of environmental stress.When stress is present, we propose that U2AF1 S34F confers a 9 survival advantage over U2AF1 WT cells, which allows for continued proliferation and accumulation of stronger oncogenic drivers like KRAS G12V , which synergize with U2AF1 S34F to increase oncogenic potential.
More work is left to be done to understand the role of splicing in stress response.For instance, the persistence of cassette exon events in U2AF1 S34F + KRAS G12V cells highlights this category of splicing events as an interesting candidate for further functional study.Intron retention (IR) is another splicing event type linked to stress response in eukaryotes.In yeast, IR has been linked to fitness advantage in the presence of environmental stressors like starvation 47 .In mouse cells, IR has been linked to osmotic stress 48 .Although we did not find evidence of altered intron retention in our short-read analysis, previous work from our group utilizing long-read sequence analysis has shown U2AF1 S34F increasing IR from long-read data 17 , leaving an interesting avenue to pursue as long-read sequencing technologies improve.
Overall, our study points to the importance of interrogating the function of cancer mutations in the context of other mutational contexts and environmental conditions to obtain a more complete understanding of their contributions to tumorigenesis.

Lead contact
Please direct all manuscript questions to Dr. Angela Brooks (anbrooks@ucsc.edu)

Material availability
Cell lines generated from this study are available upon request.10 selection to generate a stable expression of KRAS G12V or LACZ using plasmids obtained as gifts from the laboratory of John D Minna (Hamon Center for Therapeutic Oncology Research, The University of Texas Southwestern Medical Center) and used as described in Sato et al. 25 .Cell lines generated were tested for mycoplasma (IDEXX).
Mouse xenograft of HBEC3kts: Clone 1 and clone 2 HBEC3kt lines were cultured as previously described 15 .Cells were allowed to recover from cold storage in liquid nitrogen after seeding for one passage in a T-25 flask.Cells were passaged to a 10cm plate, then to a final 15cm plate, and allowed to grow to 80% confluency.At 80% confluency, the media in the 15cm plates were aspirated, and cells were washed twice with DPBS.To suspend cells for injection, cells were trypsinized with standard protocols 49 , and live cell counts were assessed by Trypan Blue staining.For each cell line, 9 million cells were resuspended in Keratinocyte SFM media containing 40% Matrigel and subcutaneously injected into the fourth abdominal fat pads on both sides of male NSG mice.2-5 million cells were injected at each site in 100 uL media + Matrigel (5 million in 1st xenograft experiment, 2 million in 2nd).Mice were monitored every week for tumor growth.All mice were euthanized if tumor growth reached end point (1500 mm 3 ), the tumors were ulcerated, or mice showed signs of distress.Tumor size was measured using digital calipers.A total of 4 female and 14 male mice were used.

RNA extraction of HBEC3kts:
For RNA sequencing of HBEC3kt lines, cells were allowed to recover from cold storage in liquid nitrogen after seeding for one passage in a T-25 flask.Cells were passaged at 70-80% confluency to maintain log-phase growth into a 10cm plate.Once cells in the 10cm plates reached 70-80% confluency, they were washed twice in ice-cold DPBS and then collected in Tri-reagent for storage at -80 o C until the bulk RNA was extracted using Direct-Zol RNA Miniprep Kit (Cat#R2050, Zymo Research).

Short-read RNA-Seq of U2AF1 WT + LACZ, U2AF1 S34F + LACZ, U2AF1
WT + KRAS G12V , and U2AF1 S34F + KRAS G12V HBEC3kts: For Illumina sequencing, n=3 10cm plates per HBEC3kt genotype of both clones, for a combined n=6 per genotype, were cultured for RNA extraction as described above.Concentrations of purified RNA in nuclease-free water were determined by Nanodrop-2000 Spectrophotometer and Qubit RNA BR Assay (ThermoFisher Scientific).RINe numbers ranging from 7.8-10 were determined by TapeStation 4150 RNA ScreenTape Analysis (Agilent Technologies) before sending RNA to UC Davis DNA Technologies and Expression Analysis Core Laboratory for poly-A strand specific library preparation to obtain 60 million paired end read pairs by NovaSeq S4 (PE150) sequencing.
Viability assay: HBEC3kts of differing genotypes were seeded in a 96 well-plate in triplicate and grown in supplemented KSFM.At multiple time points, (0, 4, and 6 days), cells were rinsed twice with DPBS, CellTiter-Glo (Cat# PRG7572, Promega) reagent was added, and cells were transferred to white opaque 96 well-plates for luminescence measurement.Luminescence at each timepoint was quantified using the VarioSkan platereader (Thermo Scientific) and normalized to the average relative luminescence units (RLU) of the 0 day timepoint.
Western blot analysis: Cell lines were cultured to 85% confluency in 10cm plates.After preparation of protein lysates in 1ml of RIPA buffer supplemented with protease inhibitor cocktail (Cat# 5892970001, Roche Molecular Systems, Inc, USA) proteins were denatured using standard denaturation techniques in beta mercaptoethanol laemmli buffer, and 15ug of denatured protein lysate was separated on a 4-15% Mini-Protean TGX Precast Protein Gel (Cat# 4561086, Bio-Rad Laboratories, Inc. USA).After transfer to 0.2 um PVDF membrane using TransBlot Turbo Transfer system (Cat# 1704272, BioRad Laboratories), membranes were incubated shaking at room temperature in 5% milk block in 1x PBST followed by incubation in KRAS G12V primary antibody at 1:250 dilution (Cat# 14412, Cell Signaling Technologies) and B actin conjugated to HRP at 1:500 dilution (Cat# sc-47778 HRP, Santa Cruz Biotechnology) in milk block overnight on an orbital shaker at 4 o C.The next day, blots were washed in PBST and incubated with secondary HRP-conjugated antibody (Cat# 7074, Cell Signaling Technologies) at 1:1000 dilution at room temperature for 1h.After washing in PBST, bands were detected using WesternSure PREMIUM Chemiluminescent substrate (Cat# 926-95000, Li-COR Biosciences) and visualized on a C-Digit Blot Scanner (Li-COR Biosciences).

Secreted cytokine analysis:
Growth triplicates of each cell line were seeded in 6 well plates and cultured with standard protocols described above to 85% confluency.Conditioned media (3 mL) above the cells was collected and cell debris spun out at 3000 x g for 10 mins at 4 o C and supernatent was stored in -80 o C before sending to Eve Technologies (Calgary, Canada) for the Human High Sensitivity T-Cell Discovery Array 14-plex (HDHSTC14) assay.Data was plotted and significance was calculated with a Mann-Whitney test on GraphPad Prism.
Proliferation immunofluorescent assays: Cell staining was performed at UCSC's Chemical Screening Center, using the BioTek EL406 with peri/syringe/wash modules for automated washing and dispensing of reagents.Cells were cultured as previously described in opticalbottom black opaque 96 well-plates (Cat# 3904, Corning).The plate was taken to the Chemical Screening Center and incubated with EdU for 1 hour at 37 o C and 5% CO2.Following EdU incorporation, cells were fixed with 5% formaldehyde (Cat# F79-500, Fisher) in basal media (Cat# PCS-300-030, ATCC) for 30 minutes at 37 o C and 5% CO2.Cells were blocked with 2% BSA in PBST for 20-60min in the dark at room temperature.Following blocking, click reagent (15ml 100mM Tris pH7.4,0.6ml 100mM CuSO4, 155.5ul 200mg/ml Na Ascorbate, 15.5ul 10mg/ml Rhodamine-Azide) was added to the cells to visualize EdU incorporation and cells were incubated in the dark for 1h at room temperature.Following azide incorporation, cells were stained with Hoechst (2.5uL in 2% BSA) to visualize nuclei and incubated in the dark for 2h at room temperature.Cells were then incubated with a primary antibody for PHH3 (Ser10) Recombinant Rabbit Monoclonal Antibody (9H12L10) (Cat# 701258, Invitrogen) at 1:5000 dilution in PBS and BSA, followed by incubation in a chicken anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647 secondary antibody (Cat# A-21443, Invitrogen) at 1:1000 dilution.
Immunofluorescent imaging and analysis: Imaging and quantification of EdU, Hoechst, and PHH3 immunofluorescent signal was performed with the Perkin Elmer Opera Phenix Plus and Harmony bioinformatics software at UCSC's Chemical Screening Center.Valid cells were 12 identified as nonborder objects with the presence of nuclear Hoechst staining.The mean EdU and PHH3 intensities of each valid object were divided by the mean Hoechst intensities to normalize for cell density.N = 12 wells in a 96 well-plate were seeded per genotype and at least 36,000 objects were analyzed per genotype.The resulting values were plotted and statistical analysis was performed on Python.v3.7.7 and Jupyter notebook v6.3.0.Significance was calculated using a Kruskal-Wallis test with Dunn's multiple comparisons test.
Growth in Low Attachment (GILA): Cells were grown to 85% confluency on regular tissue culture-treated 6-well plates, harvested by trypsinization, filtered over a nylon 70um mesh and seeded in triplicate in KSFM media at 2500 cells per well in Ultra-low attachment 96 well plates (Cat# 3474, Corning) and time points were collected for viability assays over an 14-day period.An early time point was collected at the time of seeding and used for normalization.Viability was assayed using CellTiterGlo according to manufacturer's instructions (Cat# G7570, Promega) and luminescence was measured on a VarioScan LUX plate reader (ThermoFisher).Significance was calculated using a Kruskal-Wallis test with Dunn's multiple comparisons test using GraphPad Prism.
Clonogenicity assay: Colony formation was assessed by seeding the cells in triplicate at 200 cells per 10cm plate and cultured under normal conditions, except that media was changed only twice over a 10 day period so as not to disturb colony formation.Cells were fixed in 100% methanol for 20 mins and stained in 0.5% Crystal Violet in 25% methanol for 5 mins before drying and photographing.Colonies of approximately 2mm or larger were counted in 4 separate quadrants of each plate.Significance was calculated using a Kruskal-Wallis test with Dunn's multiple comparisons test using GraphPad Prism.
Wound healing assay: HBEC3kts were seeded in 6-well plates.A 200uL pipettor and filter tip was used to create the wound in a confluent monolayer of cells.The wound was imaged at 0 and 3 hours.The number of cells that had migrated into the wound between the two time points was counted.Significance was calculated using a Kruskal-Wallis test with Dunn's multiple comparisons test using GraphPad Prism.
Cigarette smoke treatment: CSC was obtained from Murty Pharmaceuticals (Cat# nc1560725).HBEC3kts from clone 1 and clone 2 were seeded in a 96 well-plate and grown to 50% confluency.Cells were then treated with 0, 15, 60, and 120ug/mL CSC for three days.Following treatment, the cells were washed twice with DPBS and assayed using CelTiterGlo as described above.Luminescence was normalized to the 0ug/mL control.For each genotype, data was combined from n = 4 of 2 clones, for a total n of 8. Significance was calculated using a Mann-Whitney test using GraphPad Prism.
RNA-Seq Data Analysis: Raw sequencing reads in fastq files were aligned to a version of the human genome hg38 that has a region of repeats masked to make the U2AF1 locus alignable 50 , using STAR.v2.7.3a 51 with the parameters --outSAMtype BAM SortedByCoordinate --twopassMode Basic --quantMode GeneCounts --bamRemoveDuplicatesType UniqueIdentical and the Gencode v33 primary assembly gtf file.Aligned BAM files were indexed with Samtools.v1.10 52 .Mapped reads in BAM files were counted with HTSeq.v0.12.4 53 for all the annotated genes in gencode.v33.primary_assembly.annotation.gtfwith -stranded = reverse and nonunique=none parameters.U2AF1 S34F mRNA ratio: Aligned reads from clone 1 and clone 2 HBEC3kts were loaded onto the Integrative Genomics Viewer 54 (IGV) at the U2AF1 S34F mutational locus.The fraction of A (mutant) nucleotides at this locus obtained from IGV was plotted.Significance was calculated with a Mann-Whitney test on GraphPad Prism.

Differential expression analysis:
Differential expression analysis was performed with DESeq2 v1.40.2 55 on R v4.3.1 on aligned RNA sequences from clone 1 and clone 2 of our HBEC3kt lines.Gene counts were normalized and a likelihood ratio test calculation was performed to account for batch differences between samples from clone 1 and clone 2. Statistical analysis was performed on expression differences in the following pairwise comparisons: U2AF1 S34F + LacZ vs. U2AF1 WT + LacZ, U2AF1 WT + KRAS G12V vs. U2AF1 WT + LacZ, and U2AF1 S34F + KRAS G12V vs. U2AF1 WT + LacZ.
Normalized gene count comparisons: Normalized gene counts for U2AF1 and KRAS were obtained using DESeq2 for each pairwise comparison and plotted with Python.v3.7.7 and Jupyter notebook v6.3.0.Significance was calculated using Kruskal-Wallis test with Dunn's multiple comparisons test using Python's scikit_posthocs module.
JuncBASE count files were statistically analyzed with the compareSampleSets.pymodule, using the following non-default commands: --mt_correction BH --which_test t-test --delta_thresh 10.0.U2AF1 WT +LacZ samples were compared with the mutant genotypes.Then, redundant splicing events were filtered out using the JuncBASE script makeNonRedundantAS.py.To compare splicing event type distributions between the genotypes, splicing events were filtered for padj < 0.25 and abs(∆PSI) ≥ 10.We also filtered out junction-only alternative acceptor and alternative donor events, as these events have less read support than other categories.
Additionally, we filtered for intron retention events that consisted of known junctions.Statistical reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.S1, Table S2.S1, Data S1.S1, Table S3.S1.

Figure 1
Figure 1 KRAS G12V suppresses the effect of U2AF1 S34F on the transcriptome while altering gene expression in oncogenic pathways.(A) Distribution of KRAS, EGFR, and other mutations in lung ADC patients with and without mutations in U2AF1.(B) Experimental pipeline for study.Illumina RNA sequencing was performed on HBEC3kt lines with U2AF1 S34F alone, KRAS G12V alone, co-occurring U2AF1 S34F and KRAS G12V , and a wild-type control.Phenotypic assays for oncogenic phenotypes were also performed.(C) Heatmap of gene enrichment scores for gene sets differentially expressed between each genotype and the wild-type control.(D) U2AF1 S34F mRNA fraction in HBEC3kt lines with differing mutational backgrounds.Bars represent mean U2AF1 S34F mRNA fraction.(E) Distribution of KRAS mutations in lung ADC patients observed to display quasi-WT or typical-S34F expression patterns.Each box represents a single patient.** P ≤ 0.01, **** P ≤ 0.0001.See also Figures S1-S2, TableS1, TableS2.

Figure 3
Figure 3 Co-occurring U2AF1 S34F and KRAS G12V mutations increase oncogenic potential and proliferation.(A) Secreted cytokine measurements of each genotype, normalized to U2AF1 WT + LACZ levels.Bars represent mean normalized cytokine concentration.(B) Mean EdU intensity of cells, normalized by cell density.(C) Mean PHH3 intensity, normalized by cell density.Yellow lines correspond to the median of the wild-type control.The middle line in the body of each boxplot represent medians of each genotype, box limits represent quartiles, and whiskers represent the range of the most extreme, non-outlier data points.(D) Viability in low-attachment vessel for each HBEC3kt genotype.Relative viability is calculated by dividing the viability for each genotype at a certain time point, by the genotype's viability at day 0. Bars represent mean viability.(E) Top, representative injection site images of mouse xenografts.Bottom, tumor formation quantification for each HBEC3kt genotype injected.* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.See also Figure S3B-E, TableS1, TableS3.

Figure 4
Figure 4 Altered splicing in stress granule genes in U2AF1 S34F HBEC3kts is associated with enhanced stress response.(A) Splicing alteration distribution in lung ADC patients with smoking histories.Blue dots represent patients with U2AF1 mutations.(B) Viability in cigarette smoke concentrate (CSC).Concentrations are in ug/mL CSC.Bars show mean viability of each cell line.(C) Working model for U2AF1 S34F 's role in priming cells for oncogenic transformation.** P ≤ 0.01, *** P ≤ 0.001.See also Data S2, TableS1.